The present invention relates to a new process for the preparation of D-asparagine derivatives of formula I 
wherein R1 is an amino protecting group and R2 is an alkyl, a substituted alkyl or a group of formula A
R3(OCH2CH2)nxe2x80x94xe2x80x83xe2x80x83A
wherein R3 is hydrogen or a lower alkyl group and n is 1, 2 or 3.
Compounds of formula I are known. The compound N-benzyloxycarbonyl-D-asparaginemethylester is described in J. Liq. Chrom. (1994), 17 (13), 2759. A chemical synthesis of this compound is described in Tetrahedron (1997), 53 (6), 2075, where it is accomplished by a two-step reaction starting with already chiral (S)-asparagine. In a similar way, as described in Tetrahedron Asymmetry (1992), 3 (10), 1239, N-protected (S)-asparagine methyl ester is prepared in a two-step reaction, starting with already chiral N-protected (S)-asparagine. In J. Chem. Soc. Perkin Trans 1 (1983), 2287 the synthesis of the above-mentioned compound is described, starting also with a chiral reactant.
In EP 0 950 706 and in EP 0 896 057 the production and purification of novel D-aminoacylases from a microorganism belonging to the genus Sebekia or Amycolatopsis, respectively, is described. The enzymes are useful for the industrial production of chiral D-amino acids starting with racemic N-acetyl-D,L-amino acids. The D-aminoacylase (genus Sebekia) has an activity towards N-acetyl-D-asparagine of 1.4% as compared to N-acetyl-D-methionine (100%). The D-aminoacylase (genus Amycolatopsis) has a specific activity towards N-acetyl-D-asparagine of 19% as compared to N-acetyl-D-methionine (100%).
In Agric. Biol. Chem. (1987), 51 (3), 721 and in DE 2825245 the enzymatic preparation of D-amino acids, starting with D,L-5-substituted hydantoin, is described. The synthesis of D-asparagine is accomplished in a two-step reaction ( first: ring opening of D,L-5-substituted hydantoins to D-N-carbamyl amino acids by D-hydantoin hydrolase, followed second by the cleavage of N-carbamyl-D-amino acids to D-amino acids by N-carbamyl-D-amino acid hydrolase using Genus Pseudomonas AJ-1122) or in a one-step reaction using Genus Pseudomonas, Achromobacter, Alcaligenes, Maraxella, Paracoccus or Arthrobacter.
No technical enzymatic reaction for the preparation of asparagine ester derivatives has been described in the literature. This might be due to the ease of racemization and degradation of asparagine derivatives at the conventional pH-values (7-8.5) used for hydrolase reactions. A rapid inactivation of the enzymes was observed when higher, technical, more relevant substrate concentrations were used.
The present invention provides a process for the preparation of D-asparagine derivatives of formula I 
wherein R1 is an amino protecting group and R2 is an alkyl, a substituted alkyl or a group of formula A
R3(OCH2CH2)nxe2x80x94xe2x80x83xe2x80x83A
wherein R3 is hydrogen or a lower alkyl group and n is 1, 2 or 3, comprising:
a) reacting a compound of formula II 
wherein R1 and R2 are as defined above, with a protease in an aqueous solution at a pH of 6.0-7.5 and an organic solvent, and
b) extracting the D-asparagine derivative of formula I.
The present invention also provides a process for the preparation of N-protected L-asparagine of formula III 
wherein R1 is an amino protecting group and R2 is an alkyl, a substituted alkyl or a group of formula A
R3(OCH2CH2)nxe2x80x94xe2x80x83xe2x80x83A
wherein R3 is hydrogen or a lower alkyl group and n is 1, 2 or 3, comprising:
a) reacting a compound of formula II 
wherein R1 and R2 are as defined above, with a protease in an aqueous solution at a pH of from 6.0-7.5 and an organic solvent,
b) extracting a D-asparagine derivative of formula I; and
c) treating the aqueous layer from the extraction of step b) to obtain the N-protected L-asparagine of formula III.
Surprisingly, it has now been found, that enzyme inactivation during the preparation of asparagine ester derivatives could be overcome by employing an organic solvent. (The effect of the solvent is not that of a xe2x80x9csubstrate solubilizerxe2x80x9d since if it is added after the reaction has come to a stop, the reaction does not resume.) The compounds of formula I can be prepared in an improved way by the process of the present invention. The process of the invention provides for the preparation of D-asparagine derivatives of formula I 
wherein R1 is an amino protecting group and R2 is an alkyl, a substituted alkyl or a group of formula A
R3(OCH2CH2)nxe2x80x94xe2x80x83xe2x80x83A
wherein R3 is hydrogen or a lower alkyl group and n is 1, 2 or 3, which process comprises reacting a compound of formula II 
wherein R1 and R2 are as defined above, with a protease in an aqueous system, i.e., an aqueous solution, at a pH of 6.0-7.5 and an organic solvent, and subsequent extraction of the enantiomeric pure product of formula I.
In the structural formulae presented herein a wedged bond () denotes that the substituent is above the plane of the paper.
In the structural formulae presented herein a dotted bond () denotes that the substituent is below the plane of the paper.
The term xe2x80x9camino protecting groupxe2x80x9d as used herein refers to groups such as those employed in peptide chemistry as described in Green, T., Protective Groups in Organic Synthesis, Chapter 5, John Wiley and Sons, Inc. (1981), pp. 218-287, such as an allyloxy-carbonyl group (ALLOC), a lower alkoxycarbonyl group (e.g. tert.-butoxycarbonyl (t-BOC)), a substituted lower alkoxycarbonyl group (e.g. trichloroethoxycarbonyl), an optionally substituted aryloxycarbonyl group (e.g. p-nitrobenzyloxycarbonyl, benzyloxycarbonyl (Z) or phenyloxycarbonyl), an arylalkyl group (e.g. triphenylmethyl (trityl), benzhydryl or benzyl), an alkanoyl group (e.g. formyl, acetyl), an aroyl group (e.g. benzoyl), a halogen-alkanoyl group (e.g. trifluoroacetyl) or a silyl protective group (e.g. tert.-butyldimethylsilyl).
Preferred amino protecting groups are benzyloxycarbonyl, tert.-butoxycarbonyl, allyloxycarbonyl or benzoyl, especially preferred amino protecting group are benzyloxycarbonyl or benzoyl.
The term xe2x80x9calkylxe2x80x9d as used herein denotes straight or branched chain hydrocarbon residues containing 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, 1-sec-butyl, isobutyl, tert.-butyl, pentyl, hexyl, heptyl, octyl, including their different isomers. Preferably, the term xe2x80x9calkylxe2x80x9d denotes an optionally substituted straight or branched chain hydrocarbon residue containing 1 to 5 carbon atoms.
Alkyl in R2 is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, 1-sec-butyl, isobutyl or pentyl and more preferred methyl or ethyl.
The term xe2x80x9clower alkylxe2x80x9d as used herein denotes straight or branched chain hydrocarbon residues containing 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, 1-sec-butyl, isobutyl or tert.-butyl.
The term xe2x80x9csubstituted alkylxe2x80x9d as used herein denotes a straight or branched chain hydrocarbon residues containing 1 to 8 carbon atoms in which one or more hydrogen atoms are substituted by one or more hydroxy groups, lower alkoxy groups, cycloalkyl groups, aryl groups or by one or more halogen atoms. Examples are 3-hydroxybutyl, 4-methoxybutyl, 3-ethoxypropyl, 3-cyclohexylpropyl, benzyl, 2-phenylethyl, 1-fluoromethyl, 2-chloroethyl, 2,2-dichloroethyl, 3-bromopropyl or 2,2,2-trifluoroethyl and the like.
Substituted alkyl in R2 is preferably benzyl.
The term xe2x80x9ccycloalkylxe2x80x9d as used herein denotes a 3-6 membered saturated carbocyclic moiety, e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, preferably cyclohexyl.
The term xe2x80x9clower alkoxyxe2x80x9d as used herein denotes a straight or branched chain lower alkyl-oxy group wherein the xe2x80x9clower alkylxe2x80x9d portion is as defined above. Examples are methoxy, ethoxy, n-propyloxy, iso-propyloxy, n-butyloxy, iso-butyloxy or tert.-butyloxy. More preferred lower alkoxy groups within the invention are methoxy or ethoxy.
The term xe2x80x9carylxe2x80x9d as used herein denotes an optionally substituted phenyl group in which one or more aryl hydrogen atoms may be substituted by one or more phenyl groups, alkyl groups, lower alkoxy groups or halogenated alkyl groups. Examples for substituted phenyl groups are biphenyl, o-methylphenyl, m-methylphenyl, p-methylphenyl, o-methoxyphenyl, m-methoxyphenyl, p-methoxyphenyl, o-fluoromethylphenyl, m-fluoromethylphenyl, p-fluoro-methylphenyl, o-chloromethylphenyl, m-chloromethylphenyl, p-chloromethylphenyl, o-bromomethylphenyl, m-bromomethylphenyl or p-bromomethylphenyl.
The term xe2x80x9caryloxyxe2x80x9d signifies an aryl group as defined above which is bonded via an oxygen atom. Examples are phenyloxy, benzyloxy and the like.
The term xe2x80x9clower alkoxycarbonylxe2x80x9d denotes lower alkoxy residues attached to a carbonyl group ( greater than Cxe2x95x90O). Examples are methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, tert.-butoxycarbonyl and the like. Preferred lower alkoxycarbonyl is tert.-butoxycarbonyl.
The term xe2x80x9caryloxycarbonylxe2x80x9d denotes aryloxy residues attached to carbonyl group ( greater than Cxe2x95x90O). Examples are nitrobenzyloxycarbonyl, benzyloxycarbonyl (Z) or phenyloxycarbonyl.
Aryloxycarbonyl in R1 is nitrobenzyloxycarbonyl, benzyloxycarbonyl (Z) or phenyloxycarbonyl, more preferred benzyloxycarbonyl (Z).
By the term xe2x80x9carylalkylxe2x80x9d as used herein denotes a hydrocarbon group in which one or more alkyl hydrogen atoms are substituted by an aryl group such as trityl, benzhydryl or benzyl.
The term halogen stands for fluorine, chlorine or bromine.
The compounds of formula II may be prepared according to known methods from textbooks on organic chemistry e.g. from J. March (1992), xe2x80x9cAdvanced Organic Chemistry: Reactions, Mechanisms and Structurexe2x80x9d; 4th ed. John Wiley and Sons. For example, a N-protected D,L-asparagine derivative is esterified with the corresponding alcohol (e.g. methanol) in the presence of thionylchloride.
According to the invention compounds of formula I are prepared by the reaction of a compound of formula II, wherein R1 and R2 are as defined above, preferably, wherein R1 is benzyloxycarbonyl, tert.-butoxycarbonyl, allyloxycarbonyl or benzoyl and R2 is methyl, ethyl, n-propyl, isopropyl, n-butyl, 1-sec-butyl, isobutyl, tert.-butyl, pentyl or benzyl, with a protease in a solution at a pH of 6.0-7.5 in combination with an organic solvent. After the enzymatic hydrolysis of the L-asparagine derivative, the enantiomerically pure D-asparagine derivative of formula I is separated by extraction.
Suitable enzymes as catalysts for the reactions are proteases, preferably inexpensive bulk proteases of microbial origin. More preferred are Bacillus proteases (like Savinase from Novo Nordisk) or subtilisins e.g. subtilisin Carlsberg from Novo Nordisk (Alkalase) or from Solvay (Protease-L).
As an alternative the enzymes may be used in immobilized form.
According to the invention, the reaction is carried out in an aqueous-organic system having an aqueous solution at a pH of 6.0-7.5 and at least one organic solvent. The organic solvent(s) may be water-miscible organic solvent(s) (in a final concentration of up to 25%, preferably 10-25% (v/v)) and/or water-immiscible organic solvent(s) (in any ratio).
As to the aqueous phase, the aqueous solution can be a buffer solution known to be used for biochemical conversions, e.g., a common buffer solution such as sodium or potassium phosphate, used in a concentration of up to 1M, preferably between about 5 mM and about 50 mM. Such a buffer solution may additionally contain one of the usual salts, e.g., sodium or potassium chloride, LiSCN, Na2SO4 or a polyhydric alcohol, e.g. a sugar, in a concentration up to 1M, preferably 0.1 M. The solution has a pH from 6.0 to 7.5, preferably from 6.0 to 7.0, and especially preferred from 6.4 to 6.6.
The reaction pH ranges from 6.0 to 7.5, preferably the reaction pH ranges from 6.0 to 7.0. An especially preferred reaction pH ranges from 6.4 to 6.6.
Suitable organic solvents are technically common solvents. Examples are ethers (e.g. tetrahydrofuran (THF), dioxan or tert.-butyl methyl ether (TBME)), lower alcohols, esters (e.g. ethyl acetate), polar aprotic solvents (e.g. dimethylsulfoxide (DMSO), dimethyl-acetamide, N,N-dimethylformamide (DMF) or acetone). Preferred are water-miscible organic solvents (e.g. tetrahydrofuran (THF), dioxan, tert.-butyl methyl ether (TBME), lower alcohols, ethyl acetate or acetone).
The term xe2x80x9clower alcoholxe2x80x9d as used herein denotes straight or branched chain alkyl residues containing 1 to 8 carbon atoms with one hydroxy group, such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, tert.-butanol, pentanol, hexanol, heptanol or octanol, preferably methanol, ethanol, propanol, isopropanol, butanol, isobutanol, tert.-butanol and more preferred alcohol""s are methanol or ethanol. Most preferred alcohol is ethanol.
The substrate is suitably applied as a suspension in a 5-15% overall concentration (w/w). A more preferred overall concentration is 8-12%.
After addition of the enzyme, the pH of the reaction mixture is maintained under vigorous stirring at the selected pH-value by the controlled addition of a base. Preferred bases are aqueous NaOH or KOH solutions.
After termination of the reaction, the enantiomerically pure product of formula I is worked up conventionally by extraction of the reaction mixture with a suitable organic solvent. A preferred organic solvent is dichloromethane.
Optionally, the remaining aqueous layer could be treated to give the corresponding N-protected L-asparagine. This is achieved conventionally by acidification of the retained aqueous phase and filtering off the formed precipitate or its extraction with a suitable organic solvent.
Therefore, also part of the present invention is a process for the preparation of N-protected L-asparagine of formula III 
wherein R1 is as defined above, preferably wherein R1 is benzyloxycarbonyl, tert.-butoxycarbonyl, allyloxycarbonyl or benzoyl which process comprises
a) reacting a compound of formula II 
wherein R1 and R2 are as defined above, preferably wherein R1 is benzyloxycarbonyl, tert.-butoxycarbonyl, allyloxycarbonyl or benzoyl and R2 is methyl, ethyl, n-propyl, isopropyl, n-butyl, 1-sec-butyl, isobutyl, pentyl or benzyl with a protease, preferably with a microbial protease, more preferred with a subtilisin or with a Bacillus protease in an aqueous solution at a pH of 6.0-7.5, preferably at a pH of 6.0-7.0 and most preferred at a pH of 6.4-6.6, and an organic solvent. The more preferred organic solvent is tetrahydrofuran, dioxan, tert.-butyl methyl ether, a lower alcohol, ethyl acetate, dimethylsulfoxide, dimethylacetamide, N,N-dimethylformamide or acetone, and most preferred is tetrahydrofuran (THF), dioxan, tert.-butyl methyl ether (TBME), lower alcohols, ethyl acetate or acetone. The mixture is subsequently extracted to obtain the enantiomeric pure product of formula I and the aqueous layer from the extraction is retained; and
b) treating the retained aqueous layer to give the corresponding N-protected L-asparagine of formula III.
Optionally, in order to obtain a high chemical purity for the product of formula I or III, it can be triturated in the presence of a suitable organic solvent in which the product of formula I or III is, for reasons of stability, virtually insoluble.
The process is preferably carried out for the preparation of N-benzyloxycarbonyl-D-asparagine methyl ester and triturated in the presence of TBME.
The compounds of formula I, prepared according to the inventive process can be used for the preparation of optically active 3-aminopyrrolidine derivatives of formula IV 
wherein R1 is an amino protecting group and R is hydrogen, C1-12-alkyl, C3-8-cycloalkyl, C3-12-alkenyl, phenyl, tolyl, naphthyl, pyridine, pyrimidine, pyridazine, benzyl, preferably wherein
R1 is benzyloxycarbonyl, tert.-butoxycarbonyl, allyloxycarbonyl or benzoyl and R is hydrogen, C1-8-alkyl, C3-8-cycloalkyl, C3-8-alkenyl, phenyl, tolyl, naphthyl, pyridine, pyrimidine, pyridazine, benzyl, and most preferred wherein
R1 is benzyloxycarbonyl, tert.-butoxycarbonyl, allyloxycarbonyl or benzoyl and R is benzyl.
The reaction for the preparation of the optically active 3-aminopyrrolidine derivatives of formula IV may be carried out as described in U.S. Pat. No. 5,977,381, which is incorporated herein by reference.
The optically active 3-aminopyrrolidine derivatives of formula IV are important building blocks for the production of useful products in the chemical, agricultural and in the pharmaceutical industry. In particular they are useful for the production of antibacterial substances as for example vinylpyrrolidinone-cephalosporin derivatives as described in WO 99/65920, EP-A 0 620 225 or in EP-A 0 849 269.
In the following examples the abbreviations used have the following significations.